Christopher J. Schofield

CHAPTER 2:Introduction to Structural Studies on 2-Oxoglutarate-Dependent Oxygenases and Related Enzymes

Crystallographic studies have revealed that the Fe(ii)- and 2-oxoglutarate (2OG)-dependent oxygenases and structurally related enzymes employ a conserved double-stranded β-helix (DSBH, or jelly-roll) fold to enable oxidation of a wide range of substrates. The N- and C-terminal ends of the DSBH are modified, including by addition of α-helices and β-strands, in a 2OG oxygenase characteristic manner. In some cases inserts occur between the eight β-strands that comprise the core DSBH, most commonly between the fourth and fifth DSBH strands. The DSBH supports residues that enable binding of Fe(ii) and 2OG in a subfamily conserved manner. The single iron ion at the active site is normally relatively deeply bound and ligated by the side chains of three protein residues which form a conserved His-X-Asp/Glu⋯His motif. In some cases, e.g. the 2OG-dependent halogenases, only two iron ligands are present. The sizes of 2OG oxygenases vary considerably, from less than 300 residues, as observed in some small-molecule hydroxylases, to greater than 1000 residues, found in 2OG oxygenases involved in the regulation of protein biosynthesis. In the latter case additional discrete domains are commonly observed, some of which are related to dimerization or to determining substrate selectivity. The structures have revealed conservation in the general mode of 2OG binding, involving bidentate iron coordination and binding of the 2OG C-5 carboxylate by basic (Lys or Arg) and alcohol-bearing residues, but also differences which can be exploited in the generation of highly selective inhibitors. There is considerable variation in the modes of prime substrate binding, which can involve very substantial conformational changes. However, the topology of the DSBH and surrounding elements limits the residues that are involved in substrate binding and, in some cases, dimerization. In this chapter we provide an introduction to the structural biology of 2OG oxygenases and related DSBH enzymes.

CHAPTER 6:The Role of 2-Oxoglutarate-Dependent Oxygenases in Hypoxia Sensing

Animals respond to chronic limiting oxygen availability by activation of the hypoxia inducible factor (HIF) system. As shown by pioneering work on erythropoietin regulation, HIF is an α,β-heterodimeric transcription factor which contains basic-helix-loop-helix PAS domains that bind to hypoxia response elements associated with hundreds of human genes. Both the levels and activity of HIF isoforms are affected by their post-translational hydroxylation that is catalysed by the HIF-α hydroxylases, which are Fe(ii)- and 2-oxoglutarate (2OG)-dependent oxygenases. The HIF prolyl hydroxylases (PHDs or EGLN enzymes) catalyse C-4 trans-hydroxylation of prolyl residues in the C- and N-terminal oxygen-dependent degradation domains in HIF-α. These modifications signal for substantially increased HIF-α degradation via the proteasome system by promoting the binding of HIF-α to the von Hippel Lindau protein, which is a targeting component for a ubiquitin E3 ligase. There is accumulating evidence that the activity of the PHDs is limited by oxygen availability. Thus, it is proposed that degradation of HIF-α is limited by oxygen availability, at least in many normal circumstances, and the PHDs act as hypoxia sensors. In a second mechanism of 2OG-dependent oxygenase mediated control of HIF, factor inhibiting HIF (FIH) catalyses asparaginyl hydroxylation in the C-terminal transcriptional activation domain of HIF-α, a modification that reduces the interaction of HIF with transcriptional co-activator proteins, and so reduces the transcription of HIF target genes. Inhibition of the HIF hydroxylases leads to upregulation of HIF target gene expression. PHD inhibitors are presently in trials for the treatment of anaemia via upregulation of erythropoietin. This chapter focuses on the biochemical roles of the HIF hydroxylases in the hypoxic response in animals and it describes how the discovery of the roles of the 2OG-dependent oxygenases in signalling hypoxia has promoted work on their roles in other aspects of the regulation of protein biosynthesis, at both transcriptional and translational levels.

Chapter 5:Chemical Biology of Histone Modifications

Here we review current knowledge on the enzymes and related binding proteins that are involved in covalent modifications to histones. We begin with a brief overview of the field aimed at the non-expert, then focus on the covalent modifications themselves as catalysed by specific enzymes. We also focus on the consequences of these modifications for binding interactions with other proteins that modulate gene expression. The field is one that is rapidly evolving and we hope to convey some of the excitement that we feel about recent discoveries in histone science, particularly from a molecular perspective. Two inhibitors of histone modifying enzymes are in clinical use, with more likely to be introduced in the near future. Thus, the development of modifiers of histone biochemistry is of medicinal as well as basic interest.